Many diseases are caused by inherited or acquired modifications of proteins and polypeptides' structure. Conformational diseases arise when a constituent protein undergoes a change in size or fluctuation in shape with resultant self-association and tissue deposition, such as amyloid fibrils.
The role of protein deposition in neurodegenerative diseases has become evident in a large group of etiologically diverse diseases including Alzheimer's disease, Huntington's disease, Parkinson's disease, Creutzfeldt-Jacob disease, prion disorders and Type II diabetes. In each of the various diseases, a different endogenous protein self-assembles into highly ordered fibrillar structure. Though there is no specific sequence homology between the proteins associated with each one of these diseases, they are all thought to involve important conformational changes in proteins, usually produce β-sheet structures with a strong tendency to aggregate into water-insoluble fibrous polymers.
Parkinson's disease (PD), the second most common form of neurodegenerative diseases after Alzheimer's disease, is a devastating neurological disease without cure, affecting 1-2% of the elderly population. The neuropathological hallmarks are characterized by progressive and profound loss of neuromelanin containing dopaminergic neurons in the substantia nigra pars compacta with presence of eosinophillic, intracytoplamic, proteinaceous inclusions termed as Lewy bodies (LB) and dystrophic Lewy neuritis (LN) in surviving neurons. Among the clinical features of PD are motor impairments involving resting tremor, bradykinesia, postural instability and rigidity along with non-motoric symptoms like autonomic, cognitive and psychiatric problems. The cause of these pathological characteristics is not yet fully understood but it is believed that environmental factors as well as a genetic causation or a combination of the two might result in the abovementioned clinical syndromes. It is now known that less than 10% of the PD cases has a strict familial etiology while the majority of cases are sporadic. Among the mutations associated with familial PD, three missense mutations in the α-synuclein (α-syn) gene termed A53T, A30P and E46K have been widely characterized [Lotharius, J., and Brundin, P. (2002) Nat Rev Neurosci 3(12), 932-942].
The α-syn protein is composed of 140 amino acid residues. It is a small, highly charged, natively-unfolded protein. It was first identified as the major component of LB and LN. α-syn can be divided into three major regions: the amino terminal region containing several imperfect repeats of the sequence KTKEGV, a hydrophobic central domain called the non-amyloid component (NAC) region and the carboxy terminal characterized by its highly negatively charged amino acids. It is predominantly expressed in central nervous system (CNS) neurons, where it is localized at presynaptic terminals in close proximity to synaptic vesicles and can associate with lipid membranes by forming amphipathic α-helices.
α-syn is a family member of the synuclein proteins, along with beta synuclein (β-syn) and gamma synuclein (γ-syn). α-syn and β-syn are found primarily in brain tissues, located mainly in the pre-synaptic nerve terminals while γ-syn is primarily found in the peripheral nervous system and the retina, although it has also been found to be highly expressed in some tumor tissues, including breast, ovarian and bladder tissues.
The sequence of the three synucleins is highly conserved, especially within their N terminal domain. When comparing the sequences of α-syn and β-syn, there is a major difference within the hydrophobic central domain; β-syn, a 134 amino acids protein, is missing the NAC region of α-syn and does not aggregate to form amyloid fibrils during different stress conditions, such as free radicals or increased concentration.
In the case of various neurodegenerative diseases, an alteration of the quantitative ratio between the individual synucleins occurs to the extent that the relative proportion of alpha-synuclein is increased. It was possible to detect in vitro that beta-synuclein is able to inhibit the aggregation of alpha-synuclein in a dose-dependent manner (Hashimoto et al., Neuron 32 (2): 213-23 [2001]). Tests in cell cultures in which a disruption of the normal cell proliferation and differentiation was triggered by over-expression of alpha-synuclein also showed an advantageous action, in the therapeutic sense, of beta-synuclein, which further normalized the adhesion, survival and growth of neurites in these cultures. Mice, which are transgenic for alpha-synuclein, show an elevated production of this albumin and therefore exhibit a disrupted ratio in the amounts between alpha- and beta-synuclein. Over the course of aging, they form intraneuronal inclusion bodies that are similar to Lewy Bodies and also show progressive motor disruptions, which are comparable to the disruption of function in Parkinson's disease. If these animals with alpha-synuclein are crossed with beta-synuclein transgenic mice, which show an elevated expression of this albumin, a significantly higher level in the overall expression of the synucleins can restore a homoeostasis. As a result, the number of inclusion bodies is highly significantly reduced, and the characteristic neuronal function loss is completely prevented.
Alpha-synuclein is believed to play an especially important role in the pathology of Alzheimer's disease. This is indicated by the fact that a portion of this protein, the NACP (Non-Amyloid Component Protein) domain, could be demonstrated as part of the senile plaques (Yoshimoto et al., Proc. Natl. Acad Sci 92, 9141-5 [1995] and WO-9506407), in addition to the fact that about 70% of patients suffering from Alzheimer's disease exhibit Lewy Bodies in various areas of the brain, in which alpha-synuclein is also found (Eizo et al., Neurosci. Lett. 290 (1), 41-4 [2000]). In a transgenic mouse model, beta-amyloid increases the accumulation and the neurotoxicity of alpha-synuclein (Masliah et al., Proc. Natl. Acad. Sci. 98 (21): 12245-50 [2001]).
Beta-synuclein and in particular peptides derived therefrom for disruption of alpha-synucleic aggregates have been described. See for example, octapeptides according to WO/02/04482 and three additional peptides in WO02/04625. WO002/0020 and WO01/60794 describe the use of beta-synuclein as a whole molecule or methods that increase its expression in vivo for therapy of neurological diseases that are associated with alpha-synuclein. WO-A-01/60794 in particular also teaches the use of a peptide which corresponds to the N-terminal amino acids 1 to 15 of the beta-synuclein, for preventing the aggregation of alpha-synuclein and beta-amyloid. Likewise US2006/0036073 and US20080200397 teach shorter peptide fragments derived from the N-terminal amino acids 1 to 15 of the beta-synuclein, for preventing the binding of alpha-synuclein and beta-amyloid. U.S. 20010047032 teach aromatic compounds for the treatment of amyloidosis and alpha-synuclein fibril diseases. Windisch et al teach experiments with deletion mutants of β-syn which focused on the N-terminal amino acids 1-15 of the protein. They created a peptide library containing different variations of amino acid composition derived from this sequence of β-syn, with the specific aim of finding a peptide that can be used for therapeutic application immediately or can serve as a basis for developing peptidomimetic small molecules (Windisch et al., 2004, J Mol Neurosci 24(1), 155-165).